The selectins, E-, L-, and P-selectin, constitute a family of calcium dependent cell surface glycoproteins that play critical role in inflammation mainly through recognition of specific carbohydrate ligands, sialyl Lewis X (sLeX) and sialyl Lewis A (sLeA) (Berg E L, et. al., 1991. A carbohydrate domain common to both sialyl LeA and sialyl LeX is recognized by the endothelial cell leukocyte adhesion molecule ELAM-1. J. Biol. Chem. 266, 14869-14872). E-selectin is a calcium dependent cell surface glycoprotein, predominantly expressed in the vascular endothelium and bone-skin microvascular lining, and plays a crucial role in inflammation.
Among the selectin family, E-selectin (CD62E, ELAM-1 or LECAM-2) has been highlighted as a therapeutic target based on its unique role in inflammation. Unlike L-selectin, E-selectin is not constitutively expressed in endothelial cells, but transcriptionally induced by NF-κB and AP-1 in response to inflammatory cytokines such as IL-1 and TNF-α (Bevilacqua M. P. et al. (1987) Identification of an inducible endothelial-leukocyte adhesion molecule. Proc Natl Acad Sci USA vol. 84 (24) pp. 9238-42). Consequently, elevated E-selectin expression has been reported in many types of inflammatory diseases including diabetes, arthrosclerosis, rheumatoid arthritis, and cancer (Bevilacqua M. P. (1993) Endothelial-leukocytes adhesion molecules. Ann Rev Immunol. 11:767-804). In addition, E-selectin and their ligands have also been reported to play key role in the diapedesis of metastatic carcinoma cells including prostate (Dimitroff, C. J. et al., (2005) Identification of leukocyte E-selectin ligands, P-selectin glycoprotein ligand-1 and E-selectin ligand-1, on human metastatic prostate tumor cells. Cancer Res. 65, 5750-5760), breast (Jeschke, U. et al. (2005) Expression of sialyl Lewisx, sialyl Lewisa, E-cadherin and cathepsin-D in human breast cancer: immunohistochemical analysis in mammary carcinoma in situ, invasive carcinoma and their lymph node metastasis. Anticancer Res. 25, 1615-1622), colon (Matsumoto, S. et al. (2002) Cimetidine increases survival of colorectal cancer patients with high levels of sialyl Lewis X and sialyl Lewis A epitope expression on tumor cells. Brit. J. Cancer 86, 161-167), and lung (Inata, J. et al. (2007) Circulating KL-6/MUC1 mucin carrying sialyl Lewisa oligosaccharide is an independent prognostic factor in patients with lung adenocarcinoma. Int. J. Cancer 120, 2643-2649). More recently, E-selectin was found to be expressed constitutively in an endothelial cell lineage of the bone marrow (Sackstein, R. (2004) The bone marrow is akin to skin: HCELL and the biology of hematopoietic stem cell homing. J. Invest. Dermatol. 122, 1061-1069) and assist in the homing of prostate cancer cells and leukemic cells to the bone marrow through the binding of sLeX (Krause, D. S. et al., (2006) Requirement for CD44 in homing and engraftment of BCR-ABL-expressing leukemic stem cells. Nature Med. 12, 1175-1180; 10. Dimitroff et al. (2004) Rolling of human bone-metastatic prostate tumor cells on human bone marrow endothelium under shear flow is mediated by E-selectin. Cancer Res. 64 (15), 5261-9). Targeting E-selectin potentially offers a way to control the pathological infiltration of leukocytes and/or metastatic cancer cells and to target therapies and imaging agents to the inflamed vasculature of these tissues/cancers. To date, many efforts have been made to develop a high affinity ligand to antagonize E-selectin-mediated rolling and/or adhesion and diapedesis. E-selectin ligands such as monoclonal antibody (Bevilacqua M. P. et al. (1987) Identification of an inducible endothelial-leukocyte adhesion molecule. Proc Natl Acad Sci USA vol. 84 (24), 9238-42), peptide ligand (Martens et al. (1995). Peptides which bind to E-selectin and block neutrophil adhesion. J Biol Chem 270 (36), 21129-36), and carbohydrate ligand (Ernst and Magnani. (2009) from carbohydrate leads to glycomimetic drugs. Nature Reviews Drug Discovery 8 (8), 661-77) have shown selective binding to the inflamed vasculature in both experimental animal models and clinical trials (P. T. Chapman, et al., 1996. Use of a radiolabeled monoclonal antibody against E-selectin for imaging of endothelial activation in rheumatoid arthritis, Arthritis Rheum. 39 (8), 1371-1375; K. R. Zinn, et al., 1999. Specific targeting of activated endothelium in rat adjuvant arthritis with a 99 mTc-radiolabeled E-selectin-binding peptide, Arthritis Rheum. 42 (4), 641-649), however their medical application remains a challenge due to a lack of serum stability, low affinity, low specificity, and immunogenicity (Martens C L, et al., 1995. Peptides which bind to E-selectin and block neutrophil adhesion. J Biol Chem 270: 21129-21136; Bhushan M, et al., 2002. Anti-E-selectin is ineffective in the treatment of psoriasis: a randomized trial. Br J Dermatol 146: 824-831; Magnani J L, Ernst B, 2009. Glycomimetic drugs—a new source of therapeutic opportunities. Discov Med 8: 247-252). Therefore, identification of a novel ligand that blocks E-selectin mediated rolling and/or adhesion and diapedesis, with enhanced clinical compatibility is desirable. Furthermore, an antagonistic E-selectin ligand that blocks the initial adhesion of these cells to the endothelial surface is an attractive therapeutic approach against inflammation and cancer metastasis.
Aptamers are oligonucleic acid or peptide molecules that bind to a specific target molecule. Thiophosphate oligonucleotide aptamers (thioaptamers; TA) are a new class of ligands that structurally differ from RNA and DNA and can bind proteins at high (nM) affinity (for review see: Yang and Gorenstein, Progress in Thioaptamer Development Current Drug Targets, 2004, 5, 705-715; Marshall and Caruthers, 1993. Phosphorodithioate DNA as a potential therapeutic drug. Science. March 12; 259(5101):1564-70). TAs offer significant advantages over conventional peptide ligand or antibody due to their unique chemical and biological properties: a) high affinity to protein (see for review Marshall and Caruthers, 1993, ibid); b) nuclease resistance (Kusser, 2000. Chemically modified nucleic acid aptamers for in vitro selections: evolving evolution. Reviews in Molecular Biotechnology, 74 (1), 27-38); c) easy synthesis and chemical modification (for a review see Micklefield J., 2001. Backbone modification of nucleic acids: synthesis, structure and therapeutic applications. Curr Med Chem. 8(10), 1157-79); d) lack of immunogenicity (Monteith, D. K., et al., 1997. Immune simulation: A class effect of phosphorothioate oligodeoxynucleotides in rodents, Anti-cancer Drug Design, 12, 421-432). Recently methods have been developed for combinatorial selection of TA libraries consisting of 1014 random sequences (King, D J, et al., 1998. Novel Combinatorial Selection of Phosphorothioate Oligonucleotide Aptamers, Biochemistry, 37 (47), pp 16489-16493; King, D J, et al., 2002. Combinatorial Selection and Binding of Phosphorothioate Aptamers Targeting Human NF-κB RelA(p65) and p50, Biochemistry 41, 9696-9706; Somasunderam, A, et al., 2005. Combinatorial Selection, Inhibition and Antiviral Activity of DNA Thioaptamers Targeting the RNase H Domain of HIV-1 Reverse Transcriptase. Biochemistry, 44(30), 10388-10395) and have identified TAs that bind to a wide variety of target proteins (King et al. 2002, ibid; Somasunderam, et al. 2005, ibid; Kang, J, et al., 2008. Combinatorial selection of a single stranded DNA thioaptamer targeting TGF-beta-1 protein. Bioorg. Med. Chem. Lett. 18(6):1835-1839).
The majority of screenings of aptamers libraries utilize either full-length or fragments of recombinant proteins (Joyce G F, 1994. In vitro evolution of nucleic acids. Curr Opin Struct Biol. 4:331-6. Gold L, 1995. Oligonucleotides as research, diagnostic, and therapeutic agents. J Biol Chem. June 9; 270(23):13581-4; Osborne S E, et al., 1997. Aptamers as therapeutic and diagnostic reagents: problems and prospects. Curr Opin Chem Biol. June; 1(1):5-9; Ellington, A D., Szostak, J. W., 1992. Selection in vitro of single-stranded DNA molecules that fold into specific ligand-binding structures, Nature, 355 (6363): 850). However, the structural differences that result from the lack of post-translational modifications and possible misfolding of these recombinant proteins may preclude the identification of aptamers that would maintain their binding capabilities in a physiological environment.
E-selectin ligands including monoclonal antibodies, peptide, and carbohydrate ligands have shown selective binding to the inflamed vasculature in both experimental animal models and human clinical trials. However, medical applications of these ligands remain a challenge due to low affinity, low specificity, lack of serum stability, and immunogenicity. Therefore, there remains a need to address these problems.